Comparison of primary neoplastic cells with control cells of same lineage has not been undertaken to show signaling nodes that are of particular significance due to high signaling activity in individual cell-types of myeloid neoplasms. Dynamic signaling states can be compromised when samples are cryopreserved. Thus, phospho-flow analysis performed on fresh samples can be theoretically more informative in identifying previously unidentified signaling aberrations than analysis performed on preserved samples.
Phospho-flow assays, which have typically been performed by fluorescent flow cytometry, have limitations due to the number of colors available per analysis tube. At best, fluorescent cytometry allows 18-20 markers to be evaluated simultaneously. However, overlap of fluorescence emission spectra requires set up of compensation settings that can often be time consuming. Further, tandem dyes can break down and emit signal at a different wavelength than expected, confounding results. Most commercial instruments are capable of analyzing less than 10 antibodies/tube. Thus, evaluating lineage markers and functional intracellular (IC) markers in a single tube assay has not been feasible. Multi-tube analysis can be time consuming and has precluded precise mapping of functional activity to cell-type, in particular rare cell-types (such as leukemic stem cells, dendritic cells, clonal T cells, etc.), some of which require at least 8-9 lineage- and cell type-determining surface markers (CD3, CD11c, CD14, CD19, CD33, CD34, CD45, CD117, CD123, etc.) for accurate identification based on presence or absence of markers. Fluorescent-labeled antibodies are generally more expensive and less stable than metal-tagged antibodies.
New methods are needed for analysis of large numbers of cell surface and intracellular markers simultaneously.